P < 0.001). The established method could be used for the rapid detection of the four subgroups of avian metapneumovirus with high specificity and high sensitivity.In order to develop an appropriate method for high-throughput detection of avian metapneumovirus, a quadruple real-time reverse-transcription polymerase chain reaction assay was established with four pairs of specific primers and four specific probes based on the G or M gene of aMPV-A, aMPV-B, aMPV-C and aMPV-D. Its specificity and sensitivity were evaluated, and clinical samples were tested by the method. The results showed that all the four subgroups of avian metapneumovirus can be detected in the quadruple real-time RT-PCR assay simultaneously, with a detection limit of 100–1000 cRNA copies/reaction. The other common poultry viruses were negative. In the avian clinical sample detection, 39 out of 1920 clinical samples collected from 8 provinces were positive. Compared with published RT-PCR assays, the κ value of the quadruple real-time RT-PCR assay in 1920 avian clinical samples was 1.000 ( Avian metapneumovirus (aMPV) has been detected for a long time . aMPV caaMPV was found in China in 1999, but the subgroup was not identified. Subsequently, many researchers carried out a series of surveys on chicken infection with aMPV. In 2007 and 2008, serological tests were carried out on breeder flocks in some areas of Shandong province, and the results showed that the seropositive rate of several flocks reached 100% . In 2015The detection methods of aMPV include virus isolation, enzyme-linked immunosorbent assay (ELISA) and RT-PCR –20. ViruThis study was conducted according to the animal welfare guidelines of the World Organization for Animal Health and approved by the Animal Welfare Committee of the China Animal Health and Epidemiology Center. The approval number was DWFL202104.g at 4°C for 5 min. Viral RNA was extracted according to the manufacturer’s instructions of the cador Pathogen 96 QIAcube HT kit  and stored at −80°C.The aMPV-B positive sample was donated by Zhejiang Agriculture & Forestry University, and the aMPV-C positive sample was donated by the Shanghai Animal Disease Prevention and Control Center. Avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and infectious laryngotracheitis (ILTV) were all conserved in the China Animal Health and Epidemiology Center. The 1920 cloacal/throat double swabs were obtained from 25 live poultry markets or farms in 8 provinces of China according to a random sampling method. Live poultry markets or farms of 8 provinces in China were selected at random in 2019. The 1920 samples were collected from five animal species, including white pigeon, goose, chicken, duck, and partridge. The samples were stored in 0.5 mL phosphate-buffered saline  containing 10% glycerol at 4°C to 8°C. Viral RNA was extracted within 3 days after collection. The remaining samples were stored at −80°C. The samples were centrifuged at 12,000 pEASY–T5 Zero Cloning vector. An 870-bp fragment of the whole matrix protein (M) gene of aMPV-C was cloned into the pEASY–T5 Zero Cloning vector. Recombinant plasmid DNA was extracted using a SanPrep Column Plasmid Mini-Prep Kit .Four standard plasmids were developed based on the gene sequence of the aMPV reference strain . The fragments of the whole glycoprotein (G) genes of aMPV-A, aMPV-B and aMPV-D are 1193, 1263 and 1187, respectively. The G genes were cloned into the SpeⅠand NotⅠ) and purified with the DNA Extraction Kit  respectively. The purified DNA was used as template for in vitro transcription reactions with RiboMAXTM Large Scale RNA Production System–SP6 and T7 kit . The mixture of transcription reaction in vitro include 20 μL of 5× T7 Transcription Buffer, 7.5 μL of rNTPs , 10 μL T7 Enzyme mix, 30 μL Linear DNA template and 32.5μL Nuclease Free H2O. The transcription program was 37°C for 5 h. In order to remove the DNA template, 10 μL RQ1 RNase-Free DNase was added to the reaction system and incubated at 37°C for 15 min. Then, the miscellaneous proteins and various ions in the system were removed with the Rneasy MiNi Kit , and the cRNA was extracted and purified. The cRNA was quantified using a Thermo Scientific Multiskan GO Microplate Photometer . The RNA copy number was calculated according to the following formula: RNA copy number per μL = (6.02 × 1023 × plasmid concentration (ng/μL) × 10−9)/(RNA length × 340).The four standard plasmids were digested by enzyme  software 7.0 respectivly. Multiple primers and probes were designed for each subgroup. The design and evaluation of these primers and probes were performed using Oligo7 software . The priThe reaction condition of the quadruple real-time RT-PCR was optimized, including the concentration of primers and probes, temperature and time of annealing/extension. All primers and probes were dissolved to 10 pmol/μL. The optimum reaction concentration of primers and probes were selected by different volumes list in . Five an6–101 cRNA copies per reaction). Evaluation of the analytical sensitivity of the quadruple real-time RT-PCR method was performed by detecting each dilution in eight replicates.The analytical sensitivity of each component in the quadruple real-time RT-PCR was evaluated using a tenfold serial dilution of standard recombinant plasmids for the four subgroups . To verify the positive results by conventional PCR, the sequences of the PCR products were compared with the GenBank nucleotide database by online BLAST search at the NCBI website.In total, 1920 avian double swabs were detected and compared with the published RT-PCR assay for the four subgroups of aMPVs to evaluate the performance of the quadruple real-time RT-PCR assay. The primers for the published RT-PCR assay  are list2O to a final volume of 50.0 μL. The detection program was as follows: 42°C for 5 min and 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Cycle threshold (Ct) values were used, as Ct indicates the PCR cycle number at which the amount of amplified target reaches a fixed threshold.The quadruple real-time RT-PCR reaction system was performed according to the optimal conditions and programs. The results of the optimal conditions and programs were showed in 3 RNA copies per reaction. Six, four and four of eight replicates with 102 RNA copies per reaction tested positive in the quadruple real-time RT-PCR assay. For aMPV-C, reactions were positive from 104 copies per reaction. Five of eight replicates with 103 RNA copies per reaction tested positive in the quadruple real-time RT-PCR assay. The detection limits of the quadruple real-time RT-PCR assay at 95% were 102 RNA copies per reaction for aMPV-A, aMPV-B and aMPV-D and 103 RNA copies per reaction for aMPV-C . The detection results of the aMPV-A, aMPV-B, aMPV-C and aMPV-D standards are shown in The sequences of the quadruple real-time RT-PCR primers and TaqMan probes are listed in The quadruple real-time RT-PCR assay was positive for the plasmid of four aMPV subgroups and negative for the samples, including AIVs, NDVs, IBVs and negative controls. aMPV-B and aMPV-C positive samples also tested positive in the quadruple real-time RT-PCR assay, but they tested negative to the probes for aMPV-A and aMPV-D. Thus, the quadruple real-time RT-PCR assay demonstrated high specificity for the detection of all subgroups of aMPV .P < 0.001)  .Out of 1920 avian clinical samples, 39 (2.03%) were positive. The geography and host distribution of the 39 positive samples were analyzed. Whenever one or more sample from a live poultry market was tested positive, it would be recorded as a positive site. 1920 avian clinical samples were collected from 25 live poultry markets. The 39 aMPV positive samples were detected in 10 live poultry markets with a group positive rate of 40% (10/25). The distribution of the 39 positive samples in each live poultry market is shown in aMPV vaccine has not been used in China. In order to prevent spreading of the disease and facilitate treatment, early detection of viral infection, especially the establishment of a rapid and accurate detection method, has received much attention.aMPV-A, aMPV-B and aMPV-C have been detected in China with various methods , 17, 23.Most real-time PCR detection technologies employ the TaqMan fluorescent probe method. Compared with the traditional SYBR Green method, this method is highly specific. In this study, an aMPV quadruple real-time RT-PCR detection method was established using TaqMan fluorescent probes, which can quickly and accurately detect aMPVs and can be used for large-scale detection of aMPV infection in poultry flocks.Subtyping of aMPV was based on G gene. The G gene sequences of all aMPV subgroups were downloaded from NCBI. Through homology comparison, it was found that the G gene sequence of aMPV-C was poorly conserved, so the more conserved M protein gene sequence was used as a template to design primers and probes for aMPV-C detection. The G genes of aMPV-A, aMPV-B and aMPV-C are more conserved, and can be used to design detection primers and probes. Therefore, the quadruple real-time RT-PCR detection method was established based on the G genes of aMPV-A, aMPV-B and aMPV-D and the M genes of aMPV-C. The aMPV detection method was developed based on quadruple real-time RT-PCR, while genotyping was performed simultaneously. Compared with RT-PCR and virus isolation, it can reduce the probability of contamination, it can yield experimental results in a short time and it has higher specificity and sensitivity. The nucleic acid samples of aMPV-B and aMPV-C were positive in the quadruple real-time RT-PCR assay. All aMPV positive samples and four standard plasmids used for the evaluation of the quadruple real-time RT-PCR assay were positive. Due to the limitation of the source of aMPV-A and aMPV-D in China, it is currently impossible to test the ability of this method to detect these two subgroups with positive virus samples. However, according to the primer/exo-probe design method and the plasmid standard detection results, the quadruple real-time RT-PCR assay is considered to be suitable for the detection of aMPV-A and aMPV-D.In order to evaluate the quadruple real-time RT-PCR assay and investigate the prevalence of aMPV in China, 1920 double swabs of live poultry trading markets in eight provinces of Shanghai, Anhui, Jiangxi, Jiangsu, Hubei, Hebei, Guangxi and Guangdong were collected. A total of 39 samples were found to be positive, including 32 aMPV-B and seven aMPV-C samples. The clinical samples tested positive by the quadruple real-time RT-PCR were also confirmed to be positive by PCR detection and sequencing. The test results showed that 28 aMPV-B positive samples were from chicken and four aMPV-B positive samples were from duck. Moreover, six duck samples and one goose sample tested positive for aMPV-C. It can be seen that aMPV-B is common in chickens in China, and aMPV-C is common in ducks. aMPV-C is often reported in geese abroad –26. In oThe quadruple real-time RT-PCR was established in this study can detect all four subgroups of aMPV simultaneously, is not time-consuming and has good specificity and high sensitivity. This assay provides a powerful tool for the detection of aMPVs, which is important for the control of aMPV.S1 TableThe plasmid of aMPV-A, aMPV-B, aMPV-C and aMPV-D were diluted series for the sensitivity tests. The number of copies per reaction was counted which could be tested positive. The minimum number of copies per reaction was considered to the sensitivity for the four subgroup of aMPV.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table4 cRNA copies per reaction) were used as templates. The mean values of CT were calculated to analyze and screen the optimal volume of primers and probes.The quadruple real-time RT-PCR reaction system was performed according to the BioScript  Co., Ltd. PrimeScriptTM One Step RT-PCR Kit instructions. The standard recombinant plasmids for the four subgroups (10(DOCX)Click here for additional data file.S4 Table2O to a final volume of 50.0 μL.The results showed that the mean values of CT were decreasing with the increasing of primer and probes volumes. However, when the volume of primers and probes increased from 1.0μL to 1.4μL, the CT values dose not decreased significantly. Therefore, considering the cost and results of the experiment, the volume of primers and probes for the four subgroups were set as 1.0μL in the 50μL reaction mixture. The reaction mixture contained the following: 25.0 μL of 2× One Step RT-PCR Buffer Ⅲ, 1.0 μL of 5 U/μL TaKaRa Ex Taq HS, 1.0 μL of PrimeScript RT Enzyme Mix Ⅱ, 1.0 μL of the primers and probes for each subgroup of aMPV (10 pmol/μL), 4.0 μL of RNA template and RNase Free dH(DOCX)Click here for additional data file.S5 TableFive annealing/extension temperature  were tested to screen the optimal annealing/extension conditions. The results showed that CT values were smaller and the sensitivity was higher than other temperatures when the annealing/extension temperature was 59°C or 60°C. In the appropriate temperature range, the specificity of primers and probes will increase with the raising of annealing/extension temperature. Therefore, the annealing/extension temperature of the quadruple real-time RT-PCR was set as 60°C.(DOCX)Click here for additional data file.S6 TableThree annealing/extension time  were tested to screen the optimal annealing/extension conditions. The results showed that there was no significant difference in the CT values when the annealing/extension time was 20s, 30s and 40s respectively. The annealing/extension time was set as 30s to prevent the influence of stability of the experiment by too short annealing/extension time. The quadruple real-time RT-PCR cycler conditions were as follows: 30 min at 50°C, 4 min at 95°C and 40 cycles of 98°C for 30 s, 60°C for 30 s and 72°C for 30 s, followed by a final extension step for 7 min at 72°C.(DOCX)Click here for additional data file.


The Clinical Dementia Rating (CDR) and Mini-Mental State Examination (MMSE) are useful screening tools for mild cognitive impairment (MCI). However, these tests require qualified in-person supervision and the CDR can take up to 60 min to complete. We developed a digital cognitive screening test (M-CogScore) that can be completed remotely in under 5 min without supervision. We set out to validate M-CogScore in head-to-head comparisons with CDR and MMSE.To ascertain the validity of the M-CogScore, we enrolled participants as healthy controls or impaired cognition, matched for age, sex, and education. Participants completed the 30-item paper MMSE Second Edition Standard Version (MMSE-2), paper CDR, and smartphone-based M-CogScore. The digital M-CogScore test is based on time-normalised scores from smartphone-adapted Stroop (M-Stroop), digit-symbols (M-Symbols), and delayed recall tests (M-Memory). We used Spearman's correlation coefficient to determine the convergent validity between M-CogScore and the 30-item MMSE-2, and non-parametric tests to determine its discriminative validity with a CDR label of normal (CDR 0) or impaired cognition (CDR 0.5 or 1). M-CogScore was further compared to MMSE-2 using area under the receiver operating characteristic curves (AUC) with corresponding optimal cut-offs.p < 0.0001) and impaired cognition on CDR . M-CogScore achieved an AUC of 0.85 , when differentiating between normal and impaired cognition, compared to an AUC of 0.78  for MMSE-2 (p = 0.21).72 participants completed all three tests. The M-CogScore correlated with both MMSE-2 (rho = 0.54, Digital screening tests such as M-CogScore are desirable to aid in rapid and remote clinical cognitive evaluations. M-CogScore was significantly correlated with established cognitive tests, including CDR and MMSE-2. M-CogScore can be taken remotely without supervision, is automatically scored, has less of a ceiling effect than the MMSE-2, and takes significantly less time to complete. Despite having important implications for patients and families, MCI is underdiagnosed in primary care. According to the Alzheimer association, only 16% of patients who are 65 years or older are screened for cognitive impairment, even though this is listed as an integral and standard component of the annual wellness visit for patients with Medicare insurance .Accessing specialised memory units for the screening of cognitive impairment has been increasingly difficult, with the COVID-19 pandemic further impacting access, especially amongst the elderly. Screening tests such as the Mini-Mental State Examination Second Edition Standard Version (MMSE-2) can take 15 min or longer to complete , 4. The The ideal remote digital solution would not require supervision, could be completed remotely, would have good test-retest reliability, take less time to complete, would have sensitivity and specificity comparable to existing tests that screen for MCI, and would have a clear and strong theoretical grounding in established neuropsychological evaluations. Such criteria are difficult to achieve for the following reasons: 1) the results of a digital solution would need to be shown to be correlated with established cognitive screening tests in healthy controls, as well as in age, sex, and education matched individuals with early cognitive impairments , 10; 2) In this study we focused on detecting early cognitive impairments using a rapid digital solution that could surmount the aforementioned difficulties. The M-CogScore is based on three shortened tests that evaluate different cognitive domains: the Stroop test that measures cognitive flexibility and ability to inhibit interference , 14, dig2.2.1We used the Mindstep remote digital cognitive screening tool (M-CogScore), which results in an automated score. The cognitive screening is based on smartphone application-adapted Stroop, digit-symbols, and memory recall tests that accounts for both accuracy and the time taken to complete the tests. The M-CogScore was administered as part of an observational research study, along with the CDR and MMSE-2, to healthy controls and to those with impaired cognition.The well-established CDR test was administered to all participants and their companions by a certified administrator. The CDR global score range 0–3) was calculated, where 0 was normal, and anything over 0 was impaired cognition . All par was calcvia an algorithm and higher scores corresponding to more severe impairment  and 55 participants with impaired cognition or MCI (CDR 0.5–1) were recruited, matched for age, sex, and level of education.All participants attended the testing site accompanied by a companion who knew them well for the CDR examination. Each participant, and their companion, gave written informed consent to participate in the study.2.2.3.All participants attended the study site in person and completed the testing over the course of one day between November 2021 and May 2022. Participants were tested in a private room by a certified administrator who was experienced in performing the CDR and MMSE-2 in participants with impaired cognition. The testing session lasted up to two hours, with breaks permitted as required.The testing session began with the administration of the CDR to the participant and their companion, to determine inclusion to either the healthy control  or impaired cognition groups . Given the sensitivity of the subject matter, the companions took part in the CDR assessment in a private room and their answers were not shared with the participant, thus providing an environment where the companions could speak freely. To prevent distraction or assistance, the companions were not permitted to be present during the participant's testing session. Any participant who scored 2 or above was classified as a screening failure and did not complete the rest of the testing procedures.The enrolled participants were administered the 30-item standard version of the second edition of the MMSE (MMSE-2) and were scored out of 30. They were also asked a series of predefined questions on their demographics, education, lifestyle, and medical history which were recorded onto a paper case report form. The participants age, sex, and education were used to ensure matching between the two groups.Finally, the participant self-administered the Mindstep app, which includes digital cognitive screening and records details of the participant's medical history, age, education, and lifestyle factors, as previously described , 24. MinThe Mindstep app was pre-downloaded onto an iPhone 10 provided at the research site for the participant to use. If a participant was not an experienced iPhone user, they were given time to familiarise themselves with how to use it, practising with another app prior to starting the testing. The Mindstep app is designed to be used without clinical supervision and therefore the participants were not given any assistance during digital testing. They were observed from a distance to ensure that they completed the testing, and the administrator recorded any additional relevant information for the researchers. The administrators were blinded to the results of the M-CogScore.All participant data were pseudonymised at the trial site after collection, then uploaded and stored in secure Amazon Web Services (AWS) servers.2.2.4.This study involved human participants and was given favourable ethical opinion by West Midlands - Solihull Research Ethics Committee (21/WM/0202). Participants and their companions gave informed consent to participate in the study before taking part. All researchers underwent Good Clinical Practice training and procedures were conducted in accordance with General Data Protection Regulation and the 1964 Declaration of Helsinki and later amendments.2.3.Stroop test that measures cognitive flexibility and ability to inhibit interference  or handwrite the answers on screen (Symbols). Participants self-administered the app with only technical guidance as necessary.The M-CogScore is based on three shortened tests that evaluate different cognitive domains: the rference , 14, digg memory , 16, and2.3.1.The delayed free recall of lists of words is frequently used to detect cognitive impairment. Several versions have been used, sometimes with fewer words to make the test quicker and easier to administer . The tesThe Mindstep Memory test (M-Memory) involved remembering eight words over two consecutive presentations, preceded by an in-app demonstration. The first presentation flashed each word on the screen for one second. After the first presentation, there was an immediate cued recall trial with four multiple choices available for each item, of which three of the words were distractors. Subsequently, there was an immediate second presentation of the eight words, again each one flashing on the screen for one second. The second presentation of the eight words was followed by the M-Stroop and M-Symbols as interference tests, before returning to recall the items. At the second cued recall trial there were eight multiple choices available for each item, with seven of these used as distractors. Neither the first nor the second cued recall trials gave feedback on the answers.M-Memory score was calculated using the total number correct out of eight (accuracy). Time was not included in the calculation to decorrelate this score from the time denominators in the other two scores (M-Stroop and M-Symbols).2.3.2.During a standard Stroop task, participants are asked to name the colour of text in which a word is written, instead of reading the text that spells a colour. The text could be either congruent or incongruent with the colour. This test generates the so-called Stroop effect, defined as a delay in reaction time between congruent and incongruent stimuli . The StrMindstep Stroop (M-Stroop) involved eighteen trials of the coloured texts: “red”, “green”, and “blue”. The user had to identify the colour from a list of three options. The colours and text were congruent in six trials, and incongruent in twelve trials. The M-Stroop score was calculated by dividing the total number correct by the time taken, a measure dependent on both accuracy and speed .2.3.3.The Symbol Digit Modalities Test is a quick and reliable neuropsychological test to assess processing speed . It consThe Mindstep Symbols (M-Symbols) test involved matching nine symbols to their corresponding number before writing the answers on screen. These were presented in three trials of three symbols. A custom neural network algorithm was used to automatically identify the handwritten number, and this was converted to a digital number and compared to the answer. The M-Symbols test score was calculated using the number of symbols correct, divided by the time taken to complete the test.2.3.4.All scores were calculated after user data had been uploaded to AWS servers. The overall M-CogScore was calculated by taking the mean of the z-scores for M-Stroop, M-Symbols, and M-Memory scores, in line with previous neuropsychological batteries that averaged the z-scores of subtests . This me2.4.The full analysis was performed on all participants for whom data for all three tests  were available. If participants were not included in the full analysis due to incomplete or missing data, this was quantified to check which tests they did not complete, and what the average scores of any completed tests were. Average ages of excluded participants were also compared with included participants.2.5.p < 0.05). Non-parametric Mann-Whitney U tests were used for comparisons with bivariate CDR data . The area under the receiver operating characteristic curves (AUC) were computed for distinguishing between normal and impaired cognition, with optimal sensitivity and specificity cut-off thresholds for MMSE-2 and M-CogScore calculated using Youden's J statistic. 10,000 bootstrapped samples with a 0.75 proportion were used to obtain the 95% confidence intervals of the AUCs. The non-parametric DeLong method was used to compare differences between AUCs (Spearman's correlation coefficient (rho) was used to assess the strength of inter-score correlations, as both MMSE-2 and M-CogScore were not normally distributed . Continuous normally distributed data were compared using t-tests, while non-Gaussian continuous data were compared using the Mann-Whitney U (MWU) non-parametric test. Shapiro-Wilk tests were used to test for normality with an alpha threshold of <0.05 suggesting the variable was not normally distributed.The difference in median scores between the two groups  were normalised to the overall range of scores separately for both MMSE-2 and M-CogScore:Statistical significance threshold was set at 0.05. Statistical analyses were performed using python v 3.9, pandas v 1.4.1, scipy v 1.6.2, matplotlib v 3.5.1, and seaborn v 0.11.2 .2.6.https://apps.apple.com/gb/app/mindsteps/id1530813748.Anonymised analysis-ready data and the statistical analyses are available upon reasonable request by contacting the corresponding author on: alijesus.alim-marvasti@nhs.net. The digital cognitive test is freely available to download from: 3.3.1.Initially, 108 participants consented, of which one later revoked consent. Two participants scored in the more severe dementia categories on the CDR global scores (CDR > 1), their data was therefore omitted as per exclusion criteria. Of the remaining 105, seven participants' M-CogScore data failed to upload to the AWS cloud due to a failed internet connection, and a further 26 skipped parts of the M-CogScore resulting in the automated scoring mechanism labelling them as incomplete without giving a total score . Therefore, a total of 72 participants were included .72 participants completed all tests . There was a clear ceiling effect of the MMSE-2 in healthy controls, but not in those with impaired cognition .M-CogScores and CDR global scores were also correlated . Again, unlike in the MMSE, there was no ceiling effect in M-CogScores in healthy controls .Receiver operating characteristic curves (ROC) for M-CogScore and MMSE-2 are shown in Cut-off thresholds for the optimal trade-off in sensitivity and specificity, to detect impaired cognition based on CDR global scores, were < 27 for MMSE-2 and < 0.21 for M-CogScore. This cut-off of 27 for the MMSE-2 gave a sensitivity of 0.65 and specificity of 0.82. The threshold of 0.21 for the M-CogScore gave a sensitivity of 0.82 with a specificity of 0.74.3.5.Of the 33 participants for whom data was not available for all three tests , 26 had an incomplete M-CogScore. Of these 26, 9 were found to have incomplete M-Stroop scores, 5 had incomplete M-Symbols scores, and 12 had incomplete M-Memory scores, because these sections of the M-CogScore had been skipped by participants. 7 participants' M-CogScore data failed to upload to the AWS cloud due to internet connection issues, and this data was unrestorable .p = 0.12 and 0.76, respectively). The 12 healthy controls scored a mean of 27.6 ± 1.5 on the MMSE-2, while the 21 with impaired cognition scored a mean of 23.7 ± 4.3. Both groups on average scored less on the MMSE-2 than their included counterparts for whom all three test scores were available, as seen in p = 0.02 for both). Overall, the proportion of participants with impaired cognition from amongst the 33 with missing data was not significantly higher than that of included participants .Of the total 33 with missing data, 12 were healthy controls (mean age 71.0 years ± 3.3) and 21 had impaired cognition . Although mean ages for both groups were slightly older than their included counterparts , neitherp = 0.02 and 0.004, respectively). Healthy controls with incomplete data had a mean age of 71 years ± 3.3 with a range of 66–74 years. The incomplete impaired group's mean age was 71.6 years ± 7.3 with a range of 53–85 years. Although individuals with incomplete data were slightly older than participants included in the main analysis .In the incomplete M-CogScore data subgroup with 26 participants, 7 were healthy controls (CDR = 0) while 19 had impaired cognition . Both the healthy controls and impaired cognition groups, on average, scored less on the MMSE-2 than their included counterparts for whom all three test scores were available .The proportion of participants with impaired cognition in the incomplete M-CogScore data subgroup  was more than in the main analysis  and discriminative validity using CDR (p < 0.0001). M-CogScore was at least as good as MMSE-2 in distinguishing between normal and impaired cognition (M-CogScore AUC = 0.85 vs. MMSE-2 = 0.78).The digital M-CogScore showed convergent validity with the established cognitive test MMSE-2  to detect impaired cognition that is comparable to established normative data , 11, 32.However, there was a disproportionate number of incomplete M-CogScore tests. Because this incomplete data pertained to individuals who had impaired cognition on CDR and lower average MMSE-2 scores, difficulty with completing the digital test was associated with cognitive impairment in t cohort. Therefore, M-CogScore may be applicable as an initial screening tool for individuals with normal or mild cognitive impairment only.4.2.p > 0.05). The performance of M-CogScore's AUC of 85% also compares favourably to the original MMSE's performance from the literature. The AUC for the original MMSE to detect MCI in the literature ranges from 67.6%   , or 74% viduals)  34). Fu. Fup > 0M-CogScore's AUC performance of 85% also compares favourably to other cognitive impairment screening tests. For example, the Montreal Cognitive Assessment (MoCA) is a freely available 30-item questionnaire, and the Addenbrooke's Cognitive Examination III (ACE-III) is a 100-item longer questionnaire. Both have been shown to detect MCI in 200 participants  with an AUC of 82% and 84% respectively . TherefoAs a cognitive screening test, a high sensitivity (82% for M-CogScore), without significant compromise in specificity 74%), is desirable to ensure early detection of cognitive decline and early recruitment to interventional studies 4%, is de, congrueSome studies have identified MMSE scores lower than 28/30 as the optimal threshold for detecting MCI , 32, OneThis study used the CDR as the benchmark. CDR is a structured, clinician or technician-rated interview that collects information from both the patient and uniquely also the caregiver or companion in order to stage the severity of cognitive impairment across six domains: memory, orientation, judgement, problem-solving, community affairs, home and hobbies, and personal care . The CDRShort cognitive tests in general have been considered inadequate to detect MCI and the usual modifications commonly involve extending test batteries which invariably take longer to complete . ExampleIn summary, the M-CogScore is a shorter digital screening test with equivalent performance to the MMSE, MoCA, and ACE, and intended for the detection of early impaired cognition through a remotely administered and automatically scored programme.4.3.Mindstep is a remote digital software that collects information on common risk factors for cognitive impairment and the M-CogScore is administered through this software , 24. MinM-CogScore is calculated as the mean of the z-score of three modified tests based on the Stroop, Digit Symbols Modality Test, and delayed recall. Although other cognitive screening tests evaluate similar cognitive domains, speed is not explicitly utilised in the scoring of CDR or MMSE and has a unique role in M-CogScore. Speed of completion directly influences two out of three of the constituent M-CogScore test scores (M-Stroop and M-Symbols). Speed and accuracy can be a proxy for cognitive function , and can4.4.Most of the incomplete data came from the M-CogScore application. While incomplete answers or skipping subsections were given a score of zero by test administrators for paper CDR and MMSE tests, the automated scoring method of M-CogScore did not ascribe a score, and therefore marked the data as incomplete. Therefore, there were more incomplete data points from the application, which may have been due to technical difficulties, perhaps related to the M-CogScore being available only on the iPhone which may be unfamiliar to some users, or even more general difficulty using technology that may come with more advanced cognitive deficits. In line with this, we found that those who failed to complete the digital M-CogScore, compared to those who completed it, were more likely to have impaired cognition on CDR screening. Additionally, we found that the MMSE-2 scores of individuals with incomplete M-CogScores were lower. Thus, M-CogScore, may not be suitable for more advanced cognitive impairment and dementia, consistent with its design as a screening tool for early cognitive impairment. Alternatively, future studies using M-CogScore could explore the role that skipping parts of tests can have on the overall score and prediction of cognitive impairment, so as to avoid data loss. This limitation could also be incorporated to M-CogScore's design to detect impaired cognition in primary care, by flagging individuals who were unable to complete the test for formal clinical screening.The M-CogScore was constructed using the mean of the z-scores of its three constituents, one of which was not normally distributed , therefoWe used CDR as the benchmark for distinguishing between normal and impaired cognition, but CDR does not objectively test cognition and its subjective scores have moderate inter-rater reliability  . HoweverAlthough participants only had technical supervision at the test centre when completing the M-CogScore, they were in a controlled environment, while it is expected that the app would be used unsupervised at home, which may affect ecological validity. An additional limitation is that M-CogScore is currently aimed for English speakers with access to the Apple iOS.5.p < 0.0001) and detects impaired cognition at least as well as the MMSE-2 (AUC 0.85 vs. MMSE 0.78). A threshold of 0.21 in M-CogScore was approximately equivalent to an MMSE-2 score of 27, giving a sensitivity of 0.82 (vs. MMSE-2 0.65) and a specificity of 0.74 (vs. MMSE-2 0.82). While the MMSE-2 had a ceiling effect, M-CogScore did not.M-CogScore is correlated with MMSE-2 (rho = 0.54, Unlike other artificial-intelligence based solutions, M-CogScore is transpicuous in its formulation as the mean of the z-scores of three modified tests based on the Stroop, Digit Symbols Modality Test, and delayed recall. M-CogScore is applicable as an initial screening tool for individuals with normal or mild cognitive impairment, with difficulty completing the test associated with cognitive impairment in our cohort.These results are particularly noteworthy because M-CogScore is independently and remotely administered, automatically scored, and can be undertaken in under 5 min on a smartphone to screen for impaired cognition. Reliable and easily understood remote digital screening is important for the early detection of cognitive impairment and recruitment to disease-modifying neurodegenerative studies. Digital technologies have the potential to expedite patients' assessment at scale . M-CogSc


Few studies suggest the use of hypnosis in weight loss may be beneficial, especially when in conjunction with other lifestyle modifications or cognitive behavioral therapy. The primary aim of this study was to determine the ability of self-hypnosis audiotape to promote weight loss by measuring its effects on the Transtheoretical Model (TTM) of change stages and processes.This study is a 3-week randomized double-blinded parallel controlled trial among adults who have overweight or obesity. The intervention group listened to a self-hypnosis audio file while the control group listened to a placebo audio file.p-value = 0.580). Gender, baseline BMD, and baseline S-weight had no effect on the association between stage change progression and self-hypnosis. The mean difference in weight at 3 weeks was −0.63 ± 0.43 kg in the hypnosis group and 0.0 ± 1.5 kg in the control group, independent t-test, p = 0.148.Forty-six participants completed the 3-week follow-up visit. There was no association between progression across stages of change and self-hypnosis  = 1.909, Self-hypnosis was not associated with a progression in the TTM’s stages of change or with weight loss after 3 weeks. As this pilot study was underpowered, further research with larger sample size and an examination of the effect of various self-hypnosis content and duration is recommended. Obesity has recently emerged as a major health concern in high-income countries and low- and middle-income countries, particularly in urban areas. Obesity and overweight are linked to increased mortality and morbidity, accounting for 4.0 million deaths and 120 million disability adjusted life years (DALYs) globally . Diet, ei.e., an altered state of consciousness) in which the person may be more receptive to positive feedback directions. There are numerous techniques for inducing a hypnotic trance, the most common being communication, particularly verbal communication .This was a 3-week double-blinded, randomized, parallel, placebo-controlled trial. Both audio files, which were distributed to participants via (a) emails sent to a random sample of the American University of Beirut and the American University of Beirut Medical Center students and staff and (b) flyers distributed in the Family Medicine clinic’s waiting areas. Recruitment occurred between 23/12/2019 and 28/2/2020. Individuals interested in participating in the study could contact the research personnel via the contact information (phone number and email address) included in the invitation emails and flyers. Following that, a 5-min phone call was made to all interested participants to determine their preliminary eligibility.Recruitment took place 2 or greater, aged 18 to 64 years, capable of providing written informed consent, fluent in English, all men, women who are not planning to become pregnant, individuals who have attempted to lose weight previously, were planning to lose weight within the next 6 months or were dissatisfied with the results of their current weight loss plan. The exclusion criteria were as follows: illiterate, individuals diagnosed with a psychotic disorder, who are currently on antipsychotic medication, pregnant women, women planning to become pregnant during the study period, and those who did not intend to lose weight within the next 6 months or are satisfied with their weight loss progress.The inclusion criteria were as follows: BMI 25 kg/mS- and P-Weight were used to assess both processes and the stages of change following the TTM . The S- The PHQ-2 questionnaire consists of two questions that assess the frequency of depressive and anhedonia symptoms. Each question was scored from 0  to 3 (nearly every day). PHQ-2 has a sensitivity of 0.86 and a specificity of 0.78 to screen for depression at a cutoff point of 3 or more .randomization.com website, (http://www.randomization.com). An independent person prepared opaque envelopes for the USBs and labeled them with serial numbers. In a separate data file, serial numbers were linked to the actual audio file (hypnosis or placebo) and were revealed after the data was collected. The participants and research assistants were blinded to which group the participant was allocated to. Each participant received a USB flash drive containing the assigned audio file and was instructed to listen to it nightly for at least 7 days using headphones in a private room and as needed for reinforcement after that. By the end of the visit, participants had received pamphlets containing information on dietary modification for weight loss. The participants were informed that hypnosis will be used in the intervention group. They were given a brief education about hypnosis, informing the participants that they can stop the recording anytime and that they are free to accept or reject any suggestions given to them. During the follow-up visit, the research assistant repeated anthropometric measurements, and administered the S- and P-weight questionnaires and a questionnaire that assessed the use of the audio file and any change in lifestyle towardweight loss. Women were asked about their pregnancy status during the follow-up visits.Participants were asked to attend a baseline 1-h visit and 20-min follow-up visit at 3 weeks. The research assistant rescreened participants for inclusion and exclusion criteria during the baseline visit. Female participants underwent a pregnancy test. The research assistant took anthropometric measurements . Weight and height were measured on the same scale , without shoes and wearing indoor clothing. Waist circumference was measured starting at the top of the hip bone, wrapping circumferentially around the body, and leveling with the belly button in a private room. Following the assessment, each participant completed a Kobo survey in a private room using their assigned code targeting sociodemographic information about their age, gender, level of education, employment status, and previous and current weight loss methods. The PHQ2 questionnaire followed this to screen for depression and the S- and P-weight tools to collect the pre-intervention stage of change. The participants were then randomly assigned to one of two study groups and were asked to listen to the assigned audio file at the baseline visit. Block randomization was generated using the p-values were set at 0.05 and power at 80%, while accounting for 20% attrition, the sample size calculation for each group is 80 using G*Power 3.1.9.7 software.The sample size was calculated with the primary outcome defined as a step-up transition in at least one stage of change as defined by the S-Weight tool. Available data on dietary counseling and physical activity in general practice showed that approximately 10–15% of participants progressed on the contemplation and action scales as defined by the transtheoretical model without any intervention . Particip-values were set at 0.05. Data were analyzed using IBM SPSS Statistics (Version 27).Descriptive statistics were used for the demographics. The primary outcome measure was a categorical variable with three options: proportion of participants who had negative progression of at least one stage, no change in the stages, and positive progression of at least one stage. Secondary outcome measures were the difference in weight, waist circumference, and P Weight processes. Chi-squared was used to determine the association between the two groups and the stages of change. An independent t-test was used to determine the association between the two groups and weight, waist circumference, and P-weight. n = 20, 43.5%) listened to the audiofile at least once daily. During the 3 weeks, the participants listened to the audiofile at home at least once daily  and 20% (n = 9) have never listened to the audio beyond the recruitment baseline visit.Sixty-five participants met the eligibility criteria and completed the initial visit. Nineteen participants dropped out at week three, leaving 46 participants who completed the initial and 3-week follow-up visit . The twop-value = 0.580)  = 1.909, = 0.580) . Gender,p = 148). The waist circumference changed by −1.2 ± 3.5 cm in the hypnosis group and by −0.72 ± 3.7 cm in the control group, leading to a −0.48 ± 1.1 change in waist circumference favoring self-hypnosis . Participants in the hypnosis group were more likely to have followed a weight loss plan (48.0%) than those in the control group (33.3%), but the difference is not statistically significant . There was no statistically significant difference in P weight items between the two groups between baseline and 3 weeks.The mean difference in weight at 3 weeks was −0.63 ± 0.43 Kg in the hypnosis group and 0.0 ± 1.5 kg in the control group, (independent t-test, The primary objective of this randomized, double-controlled trial was to determine the effect of audio self-hypnosis on the stages of change, with the primary outcome defined as a progression in at least one stage. This pilot study did not show an effect of audio self-hypnosis on the TTM’s stages of change or weight loss at 3 weeks. A narrative review of the effects of hypnosis on weight reduction revealed that most studies included a lifestyle modification component in addition to the hypnosis . This stThis study found that audio self-hypnosis had no statistically significant effect on weight loss after 3 weeks. Similarly, self-hypnosis resulted in a similar weight reduction as diabetic education in patients with diabetes type II. . Self-hyMost of the research on hypnosis and self-hypnosis is observational and was published two decades ago . This waThis pilot study did not show an effect of audio self-hypnosis on the TTM’s stages of change or weight loss at 3 weeks. Further research with a larger sample size and an examination of the effect of various self-hypnosis content and duration is recommended.10.7717/peerj.14422/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.14422/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.14422/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.14422/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.14422/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.14422/supp-6Supplemental Information 6Click here for additional data file.


Background: To date, no oral antiviral drug has proven to be beneficial in hospitalized patients with COVID-19.Methods: In this randomized, controlled, open-label, platform trial, we randomly assigned patients ≥18 years hospitalized with COVID-19 pneumonia to receive either camostat mesylate (CM) (considered standard-of-care) or lopinavir/ritonavir (LPV/RTV). The primary endpoint was time to sustained clinical improvement (≥48 h) of at least one point on the 7-category WHO scale. Secondary endpoints included length of stay (LOS), need for mechanical ventilation (MV) or death, and 29-day mortality.Results: 201 patients were included in the study (101 CM and 100 LPV/RTV) between 20 April 2020 and 14 May 2021. Mean age was 58.7 years, and 67% were male. The median time from symptom onset to randomization was 7 days (IQR 5–9). Patients in the CM group had a significantly shorter time to sustained clinical improvement  and demonstrated less progression to MV or death  and a shorter LOS . A statistically nonsignificant trend toward a lower 29-day mortality in the CM group than the LPV/RTV group  was observed.Conclusion: In patients hospitalized for COVID-19, the use of CM was associated with shorter time to clinical improvement, reduced need for MV or death, and shorter LOS than the use of LPV/RTV. Furthermore, research is needed to confirm the efficacy of CM in larger placebo-controlled trials.Systematic Review Registration: , identifier . Since naviruses . The twoaviruses .in vitro efficacy against SARS-CoV-2 , a randomized, controlled, open-label, platform trial, we randomly assigned hospitalized patients with COVID-19 to receive either LPV/RTV or camostat mesylate, which was considered standard-of-care, for up to 10 days . The primary objective was to evaluate the efficacy of the study drugs on clinically relevant outcome parameters.At the beginning of the trial, patients were randomized to one of three treatment arms: hydroxychloroquine (HCQ), with a loading dose of 200 mg two tablet bid and a maintenance dose of 200 mg one tablet bid; lopinavir/ritonavir (LPV/RTV) 200/50 mg two tablets two times daily (bid) ; or camostat mesylate 100 mg two tablet bid. Treatment with camostat mesylate was considered standard-of-care. After the results of the SOLIDARITY and RECOVERY trials  were pubvia the drug interaction checker of the University of Liverpool (https://www.covid19-druginteractions.org) and adapted if necessary.After this amendment, patients were randomized in a 1:1 ratio to receive either high-dose LPV/RTV or camostat mesylate in the main study. If patients were randomized to receive LPV/RTV, concomitant medication was controlled interactions All patients received low-molecular-weight-heparin in a prophylactic dose if no other indication for therapeutic anticoagulation  or contraindication was present. Remdesivir was allowed if deemed necessary by the treating physicians. As soon as the findings of the RECOVERY trial were available, all patients requiring oxygen support received 6 mg dexamethasone for up to 10 days.th medical department with infectious diseases and tropical medicine at the Clinic Favoriten in Vienna, Austria due to recruitment problems elsewhere. The first patient was enrolled on 20 April 2020, and the last patient was enrolled on 14 May 2021.This study was planned as a multi-center randomized, controlled, open-label, platform trial but conducted entirely at the 4In the main study, we included hospitalized COVID-19 patients ≥18 years with laboratory-confirmed  infection with SARS-CoV-2 who required supplementary oxygen (due to oxygen saturation <94% on ambient air or >3% drop in case of chronic obstructive lung disease) or radiologically confirmed SARS-CoV-2 pneumonia. Women of childbearing potential needed to be willing to use effective contraceptive methods during the study. Exclusion criteria included severe liver dysfunction , HIV infection or active viral hepatitis, anticipated discharge from hospital within 48 h, pregnancy or breastfeeding, and allergy or intolerances to the study medication. Furthermore, we excluded patients with an estimated life expectancy of <1 month .via an online tool which was provided by the medical university of Vienna and received a consecutive randomization number for the main study and a substudy if applicable. Enrollment and randomization were performed by the study team, which consisted of doctors in charge, doctors in training, or trained scientific staff. Patients, nurses, doctors, and the study team were aware of the treatment allocation in this open-label trial. All inclusion and exclusion criteria for the main and substudies and descriptions of the substudies are listed in the Each patient was randomized Only the results of the main study (LPV/RTV vs. camostat mesylate) will be reported here.The primary endpoint was time to clinical improvement which was defined as the time from randomization to a sustained improvement of at least one category on two consecutive days compared to the status at baseline. These measurements were performed using a seven-category ordinal scale. The seven categories of the World Health Organization’s proposed scale are as follows: 1) not hospitalized, no limitations on activities; 2) not hospitalized, limitation on activities; 3) hospitalized, not requiring supplemental oxygen; 4) hospitalized, requiring supplemental oxygen; 5) hospitalized, on noninvasive ventilation or high flow oxygen devices; 6) hospitalized, on invasive mechanical ventilation or ECMO; 7) death. The score was measured daily during hospitalization.Secondary outcomes included time to sustained improvement of at least two categories compared to the status at baseline measured on the WHO ordinal scale, progression to mechanical ventilation or death, length of stay, and 29-day mortality.The study and all amendments during the study period were approved by the ethics committee of the Medical University of Vienna and Vienna ethics committee. Each study center defined its own standard-of-care treatment. Camostat mesylate was considered standard-of-care at the Clinic Favoriten where all patients were recruited. All methods were carried out in accordance with the ethical principles of the Declaration of Helsinki. All patients included in the trial signed an informed consent form.α = 0.05 with a sample size of 100 per treatment group would yield a power larger than 80%. These assumptions would translate to a hazard ratio of 1.6 when assuming exponential time-to-event curves.A sample size calculation was performed based on the primary endpoint which was defined as time-to-clinical improvement for the comparisons of two groups. Based on The sample size calculation was performed using N-Query: when the sample size in each group is 100, with a total number of events required, E, of 154, an exponential maximum likelihood test of equality of survival curves with a 0.05 two-sided significance level will have 83.15% power to detect the difference between a Group 1 exponential parameter, λ₁, of 0.069 and a Group 2 exponential parameter, λ₂, of 0.043 (a constant hazard ratio of 1.6).For qualitative variables , absolute and relative frequencies will be calculated as per the treatment group. For quantitative data, mean ± standard deviation or median and the interquartile range are reported.p-values and, if appropriate, 95% confidence intervals are presented for secondary endpoints.The primary endpoint time to sustained clinical improvement is visualized by Kaplan–Meier plots. Patients who died were censored on day 29 for this analysis (this corresponds to a Fine-Gray model with competing risks). The comparisons of the two treatment arms were performed using a log rank at a two-sided level alpha of 5%. Based on the Kaplan–Meier curves, median times with interquartile ranges are reported. Additionally, simple and multiple Cox-proportional hazard models were performed for the primary endpoint. In the Cox model treatment, arm was used as a factor. In the multiple Cox regression, we adjusted for additional baseline factors such as sex and age. The analyses of all secondary endpoints are considered exploratory, and no further correction for multiplicity was performed. For time-to-event endpoints, cumulative incidence curves and log-rank tests were calculated. Therefore, unadjusted Of the 210 randomized patients, 101 received CM and 100 received LPV/RTV, 96 of whom received high-dose LPV/RTV. Nine patients received HCQ and were not included in the analysis.The mean age of the entire population was 58.6 years (SD 15.2), and 67% were male. The mean body-mass-index was 30.3 (SD 5.7). The three most common comorbidities were hypertension, diabetes mellitus, and chronic obstructive pulmonary disease.Groups were similar with respect to all baseline characteristics.The median time from symptom onset before treatment was 7 days (IQR 7–9) in both groups. The WHO score at baseline was comparable between the two groups, with 17% having a WHO score of 3 (20% LPV/RTV vs. 15% CM), 61% a WHO score of 4 (59% LPV/RTV vs. 63% CM), and 22% a WHO score of 5 (21% LPV/RTV vs. 22% CM). Thirty-three patients were treated with remdesivir. C-reactive protein, leukocyte, and lymphocyte count did not differ between groups. For further information, see p = 0.005). The simple Cox regression model also showed a significant difference , see p = 0.005)  of at least one WHO scale category was 9 days (IQR 6–12) in the CM group and 11 days (IQR 7–21) in the LPV/RTV group (log rank test 005) see .p = 0.005) but not in patients without oxygen (p = 0.43) or on high-flow oxygen (p = 0.57), see The subgroup analysis by the WHO scale at the time of randomization showed a significant effect of CM on the primary outcome in patients with low-flow oxygen , treatment with CM led to sustained clinical improvement more quickly than LPV/RTV .p = 0.021). The median time to clinical improvement was 12 days (IQR 9–16) in the CM group and 13 days (IQR 9–28) in the LPV/RTV group.When compared to patients in the LPV/RTV group, patients in the CM group had a significantly shorter time to clinical improvement within a 29-day period. This was demonstrated by improvement of at least two WHO scale categories , occurred significantly less in the CM group  . Thirteep = 0.089]. In addition, patients in the CM group had a significantly shorter length of stay   vs. 7/100 (7%), log-rank = 0.023) . For furp-value 0.001). Severe side effects were rare (5% LPV/RTV vs. 2% CM). The most common side effects were elevated liver enzymes and diarrhea. Both were more frequent in the LPV/RTV group.All adverse events with a potential relation to one of the study drugs have been reported and listed in In this randomized controlled trial (RCT), treatment with CM, which was considered standard-of-care, significantly reduced the time to sustained clinical improvement and the combined endpoint need for mechanical ventilation or death compared to LPV/RTV. There was also a trend toward lower 29-day mortality in patients treated with CM. The hospital stay for patients treated with CM was 2 days shorter.To date, there has only been one RCT published that evaluated the effect of CM in hospitalized COVID-19 patients. This was performed in Denmark. In this trial, 205 patients were randomized in a 2:1 ratio to receive either CM (200 mg tid) or placebo for 5 days. Patients had a median age of 61 years, 60% were male, and the time from symptom onset to treatment initiation was 8 days. At baseline, approximately 30% did not require oxygen and 60% were on low-flow oxygen. During the trial, approximately 40% of the patients were treated with remdesivir and 60% with dexamethasone. No significant effect on time to clinical improvement, progression to ICU admission, or mortality was observed . This isAlthough our study was not powered for subgroup analysis, a benefit of CM in the subgroup of patients with low-flow oxygen could be shown. Despite the small sample size, the trend toward reaching the primary endpoint was consistent for all subgroups see .p < 0.001) when CM (200 mg tid) was administered for 7 days. However, it is noteworthy that the study population was treated at the beginning of the pandemic, at a time at which treatment standards were different: only 20% received corticosteroids, approximately 40% were treated with tocilizumab, and almost every patient received favipiravir  which is commonly used in HIV patients . This stRS-CoV-2 , which med by us . This suFurthermore, to rule out possible toxic drug–drug interactions as a cause for a higher number of patients requiring mechanical ventilation in the LPV/RTV group, concomitant medication was evaluated prospectively in every patient. No potential interaction was found.While significantly more side effects were reported in the LPV/RTV group, the number of adverse events was high in both groups. Most side effects are, however, typical symptoms and/or complications of COVID-19 and most likely not related to the study drugs. Diarrhea and elevated liver enzymes were the most common side effect and were reported more often in the LPV/RTV group. These are well-described side effects of LPV/RTV and therefore most likely drug-related. Severe side effects were rare but occurred more frequently in the LPV/RTV group. Overall, we observed no study drug-related adverse events in the CM group.The strength of our study is that it is the first RCT that could demonstrate that CM, an oral bioavailable antiviral drug, has a positive effect on the time to clinical recovery in hospitalized patients with COVID-19. Sensitivity analyses showed that CM had the same effect on the primary outcome. CM has been used for decades in Japan, has a good safety profile, and can be administered easily. Newer oral antivirals such as molnupiravir and nirmatrelvir/ritonavir have only proven to be effective when treating outpatients with COVID-19 infections in RCTs . Further50  or immunosuppressive drugs  is initiated.


We examined health trajectories of Dutch older workers across their exit from the workforce in the 1990s, 2000s, and 2010s, testing the hypothesis that pre-post-exit health trajectories of workers with favourable and unfavourable working conditions increasingly diverged over time due to policy measures to extend working life.N = 522). Pre-post-exit trajectories were modelled using Generalized Estimating Equations with outcomes self-rated health and physical limitations and determinants physical demands, psychosocial demands, and psychosocial resources.The Longitudinal Aging Study Amsterdam includes baseline samples in 1992/1993, 2002/2003 and 2012/2013 with two 3-year follow-up waves each. Selected respondents were aged 55 years and over who exited from a paid job within the first or second 3-year interval, up to and including the statutory retirement age (Average work exit age rose from 60.7 in the 1990s to 62.9 in the 2010s. On average, self-rated health decreased somewhat over successive periods and did not show pre-post-exit change; average physical limitations increased substantially both over successive periods and from pre- to post-exit. No support is found for our hypothesis. However, regardless of work exposures, we found sharp pre-post-exit increases in physical limitations in the 2010s.Although these findings provide no support for our hypothesis of diverging health trajectories over time based on work exposure, they show that exiting at a higher age is linked to poorer pre- and post-exit health and to pre-post-exit increases in physical limitations, suggesting greater health care costs in the near future.The online version contains supplementary material available at 10.1186/s13690-022-01008-9. In many countries, policy reforms are aimed at delaying workforce exit of older workers in order to adjust to population ageing . These rHealth trajectories across the work exit transition of workers with different working conditions have not been studied extensively so far. Work exit itself does not have an unequivocal effect on health . This unThe past decades are characterised by the implementation of a series of policy measures aimed at extending working lives in many countries. In the Netherlands, measures have resulted in an increase in the average age of exit from the workforce . For worIt is an open question whether differences in pre-post-exit health trajectories remain the same over a period in which the actual retirement age has risen. A systematic review of the scarce literature on the health effects of the increase in the retirement age reports inconclusive evidence . In partThis study examines changes across three decades in health trajectories from pre- to post-exit in three cohorts of workers. We distinguish workers exposed to higher versus lower physical demands, psychosocial demands, and psychosocial resources. Because different health measures may yield different outcomes , we examThe study sample is derived from the Longitudinal Aging Study Amsterdam (LASA), an ongoing Dutch cohort study investigating physical, emotional, cognitive and social functioning in late life , 25. TheFor this study, data from the first three waves  of each cohort were used. Exit from work was determined in two successive 3-year intervals, W1-W2 and W2-W3. In the combined cohorts, 1309 respondents did paid work (≥ 1 h/week) at W1, and 730 respondents at W2. Among the working respondents, drop-out due to mortality, refusal, intraceability or unknown work status at follow-up amounted to 253 (19.3%) between W1 and W2, and 82 (11.2%) between W2 and W3. From the remaining respondents, we included those who exited by W2 or W3 and whose age at exit was not higher than their statutory retirement age. A total of 601 respondents exited from work, for 596 of whom the age at exit was known. Applying the condition of exit before or at the statutory retirement age yielded a study sample of 522 respondents.Self-rated health was measured using a single self-report question: “How is your health in general?”, with five response categories ranging from very good to poor  . PhysicaWorking conditions were assessed using the validated General Population Job-Exposure Matrix , which Respondents were asked whether they had a paid job and if applicable, when (month and year) they exited from the work force. Thus, our definition of work exit covers all reasons of exit from paid work, i.e., 1) completely retired, 2) reached the statutory retirement age, 3) partially retired but no paid job, 4) unemployed , 5) on disability benefit, 6) inactive. Age at exit was calculated based on the month and year of exit from work and the birthdate obtained from the municipal registries. Time since exit was calculated by subtracting age at exit from age at follow-up.We distinguish two time variables: historic period and within-person time. The historic periods over which the three cohorts are followed, i.e., 1992/1993–1998/1999, 2002/2003–2008/2009, and 2012/2013–2018/2019, are denoted by the 1990s, 2000s, and 2010s, respectively. The corresponding period variable has values 0, 1, and 2. Within each cohort, the variable ‘within-person time’ denotes the time from pre-exit  to post-exit .In addition to the work exit characteristics age at exit, time since exit, and reason for exit, other covariates were considered that may confound the association between historic period, working conditions, and pre-post-exit health change. Information on sex was obtained from the municipal registries. Educational level was self-reported and categorised into low (elementary education at most), middle  and high . The number of pre-exit working hours was self-reported and was included as a continuous variable. Age at exit had a non-linear association with self-rated health and therefore was categorised as 0) 55–58 years, 1) 59–63 years, and 2) 64 years through statutory retirement age.p < 0.05; for interaction terms, at p < 0.10 ).Limitations of our study include, first, the relatively low number of respondents eligible for our study sample. However, our sample was representative of the general older population. This is supported by a comparison of the average age at retirement in our sample with national data from Statistics Netherlands. At the national level, for persons aged 60–65 the average retirement age was 60.8 in the late 1990s, which compares well with our average age of 60.7 years for the 1990s, as the retirement age did not change in this decade. In the mid-2010s, the average national retirement age for this age group was 64.4 . This isSecond, the health trajectories were based on two waves, one before and one after work exit. This allowed inclusion of a maximal number of participants in each decade. By using more than two waves, we could have addressed the possibility that health trajectories differ pre- and post-exit  or are mThird, working conditions are derived from a General Population Job-Exposure Matrix (GPJEM) for workers aged 55 and over . This GPOur study provides no support for our hypothesis of diverging health trajectories based on work exposure. What our study does show clearly is that exiting at a higher age, which was at least partly induced by policy measures in the 2010s, is linked to poorer pre- and post-exit health and to pre-post-exit increases in physical limitations. The deterioration of health accompanying exit from the workforce is critical for both individuals and society. Ex-workers in poor health are less likely to participate in society through voluntary work and informal care , 49. In Additional file 1: Table S1. Pre- and post-exit characteristics of workers by exit route. Table S2. The association of pre-post exit health change with pre-exit physical demands by cohort, excluding disability exits (N=451†). Table S3. The association of pre-post exit health change with pre-exit psychosocial demands by cohort, excluding disability exits (N=451). Table S4. The association of pre-post exit health change with pre-exit psychosocial resources by cohort, excluding disability exits (N=451). Table S5. The association of pre-post exit health change with pre-exit educational level by cohort (N=522)


Vanilla planifolia Jacks. ex Andrews is the vanilla species with the most commercial and greatest economic importance. It has been used as a case study in different cryopreservation studies that involve three vitrification-based approaches: droplet-vitrification (D-V), V-cryoplate (V-Cp) and D-cryoplate (D-Cp). The aim of this study was to compare the impact of these cryogenic techniques on vegetative growth  between cryo-derived plants and in vitro-derived controls during 12 months of greenhouse growth. Genetic stability was also assessed using the inter-simple sequence repeat (ISSR) markers. There were no significant differences found in the survival and stem lengths of the in vitro-derived regenerants and cryo-derived plants. A significant increase in the number of leaves was only detected in cryo-derived plants when using the V-Cp method. The electrophoretic profiles, based on seven ISSR primers, detected low variability: 81 total bands and 27% polymorphism. This is the first report on the assessment of vegetative growth and genetic integrity in cryo-derived V. planifolia plants recovered under greenhouse conditions. Of the three cryogenic approaches, D-Cp appears to yield V. planifolia regenerants plants with more vigorous vegetative growth and a lower level of polymorphism. Future research should focus on the reproductive growth of vanilla regenerants. Vanilla planifolia Jacks. ex Andrews is the most important worldwide, as it is the natural source of the famous multi-industrial vanilla extract [Among the cultivated vanilla species,  extract .Vanilla is a monopodial and climbing orchid characterized by a single robust stem with oblong, thickened waxy leaves. Aerial roots and flower stems emerge from nodes found along the stem next to the leaves . This emV. planifolia germplasm. For instance, the cryopreservation of vanilla shoot-tips was reported for the first time in 2009 [Different biotechnological techniques, such as tissue culture ,6,7, tem in 2009  and sinc in 2009 .V. planifolia shoot-tips. Using D-V, the main problem was associated with oxidative damage [Vitrification-based procedures, such as droplet-vitrification (D-V) , V-cryope damage . Followie damage ,11. Howee damage .V. planifolia [No matter how optimized a cryogenic protocol is, the impact on the genetic stability of viable regenerants should always be verified. There are several useful markers to assess the genetic integrity of in vitro cultured and cryo-derived plants. One of the most reliable and simplest techniques is the inter-microsatellite sequencing through the ISSR (inter-simple sequence repeat) markers . These manifolia ,18.The vegetative growth studies , which cV. planifolia shoot-tips. Three cryogenic techniques were compared: D-V, V-Cp and D-Cp. The genetic stability of the cryo-derived and in vitro-derived plants was also assessed using ISSR markers.The aim of this study was to evaluate the vegetative growth  during a greenhouse culture of regenerated plants after the cryopreservation of The results after the cryopreservation of vanilla shoot-tips were very similar to that previously reported by Hernández-Ramírez et al.  and Gonzp ≤ 0.05) were found in the survival (%) between the in vitro controls and cryo-derived plants after acclimatization and the same culture period in the greenhouse  of the in vitro controls and cryo-derived plants were not significantly different regardless of the technique used . The iniA significantly higher number of leaves were recorded in cryo-derived plants when the V-Cp method was used . The higSimilar vegetative growth was observed in the cryo-derived plants and the in vitro controls after 12 months of greenhouse culture. Likewise, the leaf shape and plant color were also visually similar. However, the stems from cryo-derived (D-Cp > V-Cp > D-V) plants were slightly thicker (~up to 0.5 cm) compared to the in vitro controls .The analysis of the electrophoretic profiles, according to the seven ISSR primers , revealed a total of 81 bands (286 amplicons), ranging from 320 to 4000 bp. Of these, 59 bands were monomorphic and 22 were polymorphic, which represented 27% of total polymorphism.Of the studied ISSR primers, five  produced well-defined polymorphic bands. The main differences in banding patterns were observed in the cryo-derived plants when using the V-Cp method , and in the in vitro controls . The summary of the analysis with the ISSR primers is presented in Different studies have shown that cryopreservation results in little to no changes in the genetic stability of recovered plants ,21,22. HV. planifolia are anatomically located in the upper part of each leaf. However, after 12 months of culture, only about one new shoot per plant was detected, regardless of whether the plants were from the cryo-derived or in vitro controls (data not shown). This allowed us to visually detect that this quantitative characteristic did not induce a greater lateral growth, since apical dominance prevails and, consequently, the development of the plants is given by the elongation of the main stems, among which there were no significant differences detected. Despite the lengths being not statistically different, the cryo-derived plants had slightly thicker stems than the in vitro controls , a significant increase in the number of leaves was only detected in the cryo-derived plants when using the V-Cp method . In lighcontrols g, demonsThe increase in the number of leaves detected during the greenhouse culture in cryo-derived plants from the V-Cp protocol could be associated with a ‘hormetic effect’. According to Calabrense , the horChrysanthemum morifolium plants was less vigorous in the early stage of greenhouse culture. By contrast, Castillo et al. [Rubus plants were more vigorous than the greenhouse mother plants. On the other hand, a field performance comparison of the cryo-derived, micropropagated, and field sucker-propagated Musa plants showed similar vegetative and reproductive growth among the three plant sources [Different responses have been found after comparing the vegetative growth of the cryo-derived plants of various species. Bi et al.  found tho et al.  determin sources .V. planifolia plants for 12 months in a greenhouse is in agreement with the observations of similar studies in several species. The next step for the assessment of the vanilla regenerants will, therefore, be to compare the reproductive growth between the control and cryo-derived plants.Therefore, data regarding the vegetative growth of the cryo-derived Genetic stability has also been assessed in the cryo-derived regenerants of different plant species ,21,26. TOf the ISSR primers tested in this study, five revealed a 27% total polymorphism in the regenerants derived from the cryopreserved vanilla shoot-tips . The V-CChrysanthemum cryopreserved using the vitrification and encapsulation–dehydration methods. They found that variations occurred at low frequency (1 regenerant out of 25 showed a different band pattern) and followed the encapsulation–dehydration method only.Martín and González-Benito  detectedV. planifolia have focused on the impact of the in vitro culture techniques. Some studies have detected 71.66% polymorphism in plantlets regenerated by indirect organogenesis [V. planifolia and V. insignis shoots subjected to in vitro conservation by minimal growth.Until now, most of the molecular analyses performed on ogenesis ; others,ogenesis  found loVanilla planifolia that were propagated for 6 months after the recovery of each successive step of a droplet-vitrification protocol [By using the ISSR markers, genetic stability was also assessed in the in vitro regenerants of protocol . The cluprotocol .V. planifolia regenerants during the in vitro  and ex vitro (greenhouse) cultures detected a similar polymorphism using the same (T06) ISSR primer in both studies: D-V (22% for the in vitro and 25% for the ex vitro plants), V-Cp (25% for the in vitro and 33% for the ex vitro plants) and D-Cp (25% for the in vitro and ex vitro plants). Therefore, it is possible to speculate that, after acclimatization, no additional variations to those previously observed under the in vitro conditions were generated.A comparison of the effects of the D-V, V-Cp and D-Cp techniques on the genetic stability of the V. planifolia plants grown under greenhouse conditions and the comparison of the three cryogenic approaches focused on here. Based on the overall results, D-Cp appears to be the most recommendable cryopreservation method for V. planifolia. According to Hernández-Ramírez et al. [In summary, to the best of our knowledge, this is the first report on the evaluation of vegetative growth and genetic integrity in cryo-derived z et al. , this prz et al. , and whiz et al. .Vanilla planifolia Jacks. ex Andrews plants that were subcultured every 12 weeks on an MS [−1 6-Benzyl amino purine (BAP), 0.5 mg L−1 Indole-3-butyric acid (IBA), 20 g L−1 sucrose and 7 g L−1 agar , USA). The pH of the culture medium was adjusted to 5.7 ± 0.05 and then sterilized in an autoclave at 121 °C with 1.3 kg cm−2 of pressure for 15 min. Donor plants were maintained at 24 ± 2 °C under a 16 h light/8 h dark photoperiod with a light intensity of 36 μmol m−2 s−1 [The apical shoot tips, 3–5 mm in length, were dissected under sterile conditions from the in vitro-multiplied on an MS  semisoliThe dissected shoot tips that were used for the cryopreservation experiments were first preconditioned on a basal-MS-semisolid medium, supplemented with 0.15 M trehalose for 1d, and maintained under the same temperature and photoperiod conditions as donor plants . Afterwaw/v) glycerol, 15% (w/v) ethylene glycol, 15% (w/v) DMSO and 13.7% (w/v) sucrose) [Following D-V, the preconditioned samples were loaded with a solution containing 0.4 M sucrose + 2 M glycerol for 30 min and were then exposed to the vitrification solution, PVS2 (30% (sucrose)  for 30 mFollowing V-Cp and D-Cp, the preconditioned shoot tips were first attached with calcium alginate to the surface of aluminum cryoplates (37 mm length × 7 mm width and 0.5 mm thickness) . For encRegardless of the cryopreservation procedure used, after 1 h of storage at −196 °C, a rewarming of the samples was performed rapidly by plunging the aluminum foils and/or cryoplates into an unloading solution containing 1.2 M sucrose for 15 min. A recovery culture of shoot tips after warming was carried out in a semisolid-MS-multiplication medium in dark conditions for about 4 months until any kind of recovery was detected.−1 6-Benzyl amino purine (BAP), 0.5 mg L−1 Indole-3-butyric acid (IBA), 20 g L−1 sucrose and 7 g L−1 agar . The pH of the culture medium was adjusted to 5.7±0.05 and then sterilized in an autoclave at 121 °C with 1.3 kg cm−2 of pressure for 15 min. Two subcultures were performed in 60-day periods prior to rooting in the MS-semisolid medium without growth regulators. The cultures were exposed to 24 ± 2 °C under a 16 h light/8 h dark photoperiod with a light intensity of 36 μmol m−2 s−1.As previously described, the MS-multiplication medium was supplemented with 1 mg LAfterward, all regenerated plantlets derived from the cryopreserved shoot tips were grown in glass tubes using the basal-MS medium to promote the elongation of the aerial part until reaching approximately 10–12 cm in length and favoring the spontaneous formation of well-developed roots .V. planifolia plants during greenhouse culturing.Regenerated plants derived from the non-cryopreserved shoot tips and subjected to the same in vitro culture conditions were used as the in vitro controls for assessing the vegetative growth and genetic integrity of The ex vitro culture experiments were carried out in the greenhouses belonging to the Enterprise of Ornamental Tropical Crops of Veracruz in Cordoba city, Veracruz. The in vitro and cryo-derived 8-week-old plants were acclimatized. The in vitro and cryo-derived plants 8-week-old were acclimatized.−1 fosetyl-Al  was applied at the base of the roots. The plants were transplanted into plastic pots (11 height × 14 width) containing 250 g of sterilized 387 tezontle . The pots were covered with a transparent plastic cap and progressively opened every 4 d for 30 d before being completely removed [The culture medium was gently removed from the roots of tissue-culture plants with abundant sterile water, and 1 g L−1 Triple 20® . All the greenhouse-grown plants (the in vitro controls and cryo-derived) were carefully monitored to assess their survival (%) and vegetative growth during the 12 months of culture.After 30 d, the plants were grown in a substrate composed of 50% tezontle and 50% pine bark. Each plant was placed in the substrate 1 cm deep, taking care to cover the base of the stem and the longest roots to avoid dehydration. The aerial roots were left exposed, and a pinewood stake was placed as a supporting tutor. The plants were watered twice a week with running water and fertilized using 1 g LThe vegetative growth of the in vitro (non-cryopreserved controls) and cryo-derived plants (regenerants recovered after using the three cryopreservation protocols described) was expressed by the increase in stem length , and by the increase in the number of new leaves formed after 3, 6, 9 and 12 months. The initial stem length, of both the in vitro controls and cryo-derived plants, was 12.08 cm on average. The initial number of leaves, of both the in vitro controls and cryo-derived plants, was 5.88 on average. The survival (%) of the plants was recorded after the same growth periods were referred to as the initial number of planting plants and the number of surviving plants across the culture time. The survival (%) of the plants was recorded after the same growth periods by comparing the initial number of planting plants with the number of surviving plants across the culture time.® 17 statistical software, State College, Pennsylvania, USA through an analysis of variance (ANOVA) and the Tukey’s test with a significance level of p ≤ 0.05.Survival was analysed by evaluating 50 plants grouped into 5 replicates of 10 samples, randomly selected for both the in vitro controls and cryo-derived plants per each cryopreservation technique and culture period  in the greenhouse. The analysis of the stem lengths and the number of formed leaves was performed in triplicate, considering a total of 30 randomly selected plants for both the in vitro controls and cryo-derived plants. All data were analysed with the MinitabA general illustration of the performed studies in this work is represented in w/v) agarose gel stained with 2.53 µM ethidium bromide  and visualized in a UV light using a GelDoc-It photo-documentation system. The ISSR analysis was conducted using seven ISSR primers (Sigma-Aldrich®), as presented in The leaf samples (0.5 g) from ten randomly selected in vitro controls and cryo-derived plants after six months of culture in a greenhouse were used for DNA extraction. The extraction was performed according to a modified Doyle and Doyle method  and carrTM thermocycler, Axygen Scientific, Union City, CA, USA, under the following program: 4 min at 94 °C, followed by 35 cycles, each comprising 50 s at 94 °C for denaturation, 45 s at 49–56 °C for annealing , 90 s at 72 °C and a final extension of 10 min at 72 °C.The PCR was performed in a 25-μL reaction solution containing: Crystal Buffer  (1×), DNTP’s , Taq Polymerase , primer  and 1.0 μL of DNA (20 ng). The PCR products were amplified using a MaxyGene−1). A marker of 500 to 5000 bp was used to visualize the size of the bands. Electrophoresis was performed at 90 V for the first 20 min, followed by 100 V for 1 h, and the gel was visualized in a transilluminator with UV light.The PCR products were electrophoretically separated on 1.8% agarose gels. The gel was prepared with 1× TBE buffer and stained with ethidium bromide , V-cryoplate (V-Cp) and D-cryoplate (D-Cp).The present study focuses on evaluating the vegetative growth and genetic integrity of After systematically monitoring plant growth for 12 months in a greenhouse, there were no significant differences found in the general vegetative growth between the cryo-derived plants and the in vitro controls (non-cryopreserved plants). A significant increase in the number of leaves was only detected in the cryo-derived plants when using the V-Cp method.The genetic integrity assessments revealed 27% total polymorphism using seven ISSR primers. The V-cryoplate procedure resulted in a higher polymorphic rate compared with the D-V and D-Cp techniques and the in vitro-derived control. However, the generally low level of variation suggests that there were no changes in the genetic fidelity of cryo-derived plants.V. planifolia. Future studies will focus on the impact of these techniques on vanilla reproductive growth.Currently, this is the most advanced work on the vegetative growth and genetic integrity assessment of cryo-derived regenerants of